Translation of abstract (English)

The members of the p24-protein family constitute a highly conserved family of type I transmembrane proteins that cycle between the organelles of the early secretory pathway. They were shown to bind and recruit the components of the COPI vesicle coat, ARF1 and coatomer, in their homodimeric form and thus initiate vesicle biogenesis. Moreover, interactions between the p24-proteins and components of the COPII coat were reported. To date, however, the regulation of selective binding of COPI components to Golgi membranes and COPII components to ER membranes, respectively, has not yet been elucidated. In a series of in vitro studies, the formation of different heterooligomers was described, whereas the existence of homodimers in vivo has not yet been studied. The aim of the present work was to investigate in vivo the formation of homodimers in the Golgi as well as in the ER by utilising Fluorescence-Resonance-Energy-Transfer (FRET). Based on the determined FRET-efficiencies, p23, p24 and p25 form homodimers to a great extent in the Golgi region, whereas p27 only showed weak interactions and p26 was found exclusively in its monomeric form. These homodimers, as well as the already reported heterodimer of p23 and p24 dissociate in the ER. Moreover, fixing ARF1 in its exclusively GDP- or GTP-bound state does not have an influence on the oligomeric state of p23 and p24. These findings demonstrate how p24-proteins regulate the recruitment of ARF1 and coatomer to the Golgi membrane by exclusively forming homodimers in the Golgi. The goal of a second project was to establish a method to investigate the fate and function of COPI vesicles in vivo. To this end, fluorescently labelled COPI vesicles were generated in vitro from isolated rat liver Golgi membranes. After purification and characterisation they were microinjected into mammalian cells. Due to the fluorescently labelled vesicle proteins they could unambiguously be identified and further monitored. Within 30 minutes these vesicles moved independently of microtubules towards the Golgi and eventually fused with this compartment. Some vesicles, particularly those containing Mannosidase II, fused directly with the Golgi and not with ER membranes. The labelled, exogenous proteins behaved like endogenous proteins after BFA treatment and could be classified into two different populations. This system now provides the opportunity to more unambiguously address several open questions about the fate and function of COPI vesicles in vivo.